1. The study evaluated the pharmacognostic profile and phytochemistry of Sphaeranthus indicus leaves, which are used traditionally in Ayurveda to treat various diseases. 2. Macroscopic and microscopic analysis of the leaves was performed according to standard procedures. Physicochemical parameters and preliminary phytochemical analysis revealed the presence of sterols, terpenoids, flavonoids, and volatile oil. 3. Thin layer chromatography identified isoflavone glycosides in methanol and aqueous extracts. Gas chromatography was used to analyze and identify components of the isolated volatile oil.

1. Available online on www.ijppr.com
International Journal of Pharmacognosy and Phytochemical Research 2018; 10(1); 52-62
ISSN: 0975-4873
Research Article
*Author for Correspondence: vddpaul@gmail.com
Pharmocognostical and Phytochemical Evaluation of Leaf of
Sphaeranthus indicus
Dhanapal Venkatachalam, Samuel Thavamani b, Muddukrishniah
Department of Pharmacognosy, Sanjo College of Pharmaceutical Studies, Velappara, Palakkad, Kerala – 678 702.
Received: 19th
Dec, 17; Revised 7th
Jan, 18, Accepted: 12th
Jan, 18; Available Online:25th
Jan, 18
ABSTRACT
Objective: To study detailed Pharmacognostic profile and preliminary phytochemical investigation and isolation of volatile
oil, and TLC and GLC analysis of volatile oil of the leaves of Sphaeranthus indicus (Linn.) commonly known as Globe-
thistle belongs to the family Asteraceae. The leaves of Sphaeranthus indicus (Linn.) used traditionally in Ayurveda for
hyperlipidemia, epilepsy, mental illness, jaundice, diabetes, leprosy, fever cough, gastropathy, hernia, hemorrhoids,
helminthiasis, dyspepsia and skin diseases and AIDS. The reports showed that it is also used for hypertensive, anxiolytic,
neuroleptic, immunomodulatory, antioxidant, anti-inflammatory, bronchodilator, anti-hyperglycaemic and hepato
protective. It grows in rice fields, dry waste places and cultivated lands in tropical parts of India. Methods: Leaf of
Sphaeranthus indicus (Linn.) was studied by Macroscopical, Microscopical,Quantitative Microscopy, Physicochemical,
Phytochemical analysis of leaf powder and extracts, isolation of volatile oil from the leaf, TLC and GLC analysis of the
oil of the leaves and other methods for standardization recommended by WHO. Results: Macroscopically leaves are
simple, alternate, oblong, spatulate, spinous, surface pubescent, base decurrently forming the wings of the stem, acute,
glandular, hairy and narrowed at the base up to 5.0x1.5 cm, leaf margins are coarsely serrate or dentate. Fresh leaves are
dark green in colour and dried leaves are greenish black colour. The leaves are bitter in taste with pleasant odour when
fresh, the aroma gradually diminishing on drying and storing. The leaf has distinct midrib and thick, soft lamina. The
midrib is plano-convex in cross-sectional view with single top-shaped collateral vascular bundle surrounded by
parenchymatous cells. No sclerenchyma cells are seen in the vascular bundle. The lamina is dorsiventral; however the
mesophyll tissue is not well differentiated into palisade and spongy tissues. Characteristic epidermal trichomes are
abundant on the leaf. Some of the trichomes are covering-type and are multicellular, uniseriate, unbranched and whip-like
others are biseriate, broad, unbranched, conical with vertically oblong cells and a few tiers of apical glandular cells. Stomata
are anomocytic; anticlinal walls of the epidermal cells are highly wavy. Vein islets are distinct, with one, simple or branched
vein terminations. Petiole is circular in sectional view with aerenchymatous outer ground tissue, broad central tissue and
is open ring of discrete collateral vascular bundles. The investigations also included leaf surface data; quantitative leaf
microscopy. Physiochemical parameters such as loss on drying, extractive values and ash values were also determined.
Preliminary phytochemical screening showed the presence of sterols, terpenoids, carbohydrates, flavonoids (Isoflavone),
tannins and volatile oil. TLC studies reveal that the presence of isoflavone glycosides. Essential oil have been analysed
by GLC and their components were identified and quantified. Conclusions: The results of the study can serve as a valuable
source of information and provide suitable standards for identification of this plant material in future investigations and
applications.
Keywords: Sphaeranthus indicus, Ayurvedic system, Volatile oils, T.L.C and GLC.
INDRODUCTION
Herbal medicine is the oldest form of health care known to
mankind. Herbs had been used by all cultures throughout
history. Some are made from plant extracts; others are
synthesized to mimic a natural plant compound 1
. The
world Health organization (WHO) estimates that about 4
billion people, 80% of the world population presently use
herbal medicine for some aspect of primary health care2
.
In almost all the traditional medicine, the medicinal plants
play a major role and constitute the backbone of the
traditional medicine3
. Indian Materia Medica includes
about 2000 drugs of natural origin almost all of which are
derived from traditional system and folklore practices.
Medicinal plants are inextricably inter-twined with the rich
history, culture and culinary tradition of India. India has a
rich and glorious ethno medical heritage4
. Medicinal plants
are also used by the codified systems of medicine such as
Ayurveda, Siddha, Unani, Chinese and Tibetian systems of
medicine5
with the advent in science, many of the crude
drugs used in traditional system have been investigated
scientifically. Sphaeranthus indicus Linn. is a herbal plant
widely used in Ayurvedic system of medicine for treating
different diseases6
. It grows well in waste lands, paddy
fields, places and it is also cultivated in tropical and
subtropical parts of India. It is usually found in throughout
India, some parts of Sri Lanka, Africa and Australia from

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Table 1: Quantitative evaluation of the crude drug of
leaf of Sphaeranthus indicus.
S. No Plant constants Values
1 Vein islet no 14.4
2 Vein termination no 17.5
3 Stomatal number (upper) 18.8
4 Stomatal number (lower) 50.6
5 Stomatal index (upper) 24.5
6 Stomatal index (lower) 35.4
1.
sea level to 1200 m altitude7
Pharmacognostic studies on
leaves are not adequate necessitating the present
investigation. Though chemical analysis of the volatile oil
of capitulam of this plant was well documented with GC-
MS8
and since no detailed studies seems to have been
previously done on the leaves pertaining to the volatile oil
content and chemical analysis of the same. so the present
study is aimed to isolate and evaluate the volatile oil from
the leaves of this plant using GLC technique which is an
ideal method for both the quantitative and qualitative
analysis of the constituents of Essential oil.A novel
isoflavone glycoside have been reported on leaves of
methanolic extract of S. indicus9
. So it is planned to
prepare the methanolic extract and aqueous extract which
is subjected to preliminary phytochemical screening and
TLC studies to identify the presence of active principles.
MATERIALS AND METHODS
Plant material
Sphaeranthus indicus leaf was collected, from in and
around of Palakkad district, Kerala, India and
authenticated by taxonomist and the plant authenticated
specimen is deposited in the Department of
Pharmacognosy, Sanjo college of pharmaceutical studies,
Palakkad. Authentication specimen number is
SCPS/P.COG/002/2017 the fresh leaves were kept for
shade drying. Dried specimen was powdered using
mechanical grinder and passed through 60 mesh sieve to
get the powder of desired coarseness. Powdered material
was preserved in an air tight container.
Pharmacognostic studies
Sphaeranthus indicus (Linn) is an aromatic, annual herb.
Family: Asteraceae
Systematic position10
Phylum : Spermatophyta
Division : Angiosperms
Class : Dicotyledons
Sub class: Sympetalae
Order : Campanulales
Family : Asteraceae
Genus : Sphaeranthus
Species : Sphaeranthus indicus
Synonym: Sphaeranthus hirtus
Common Names
Figure 1 : T.S of leaf through
midrib.
Figure 2 : Vascular bundles enlarged.
Figure 3: T. S of lamina through lateral vein.
Table 2: Physico chemical evaluation of the crude drug
of leaf of Sphaeranthus indicus.
S. No Physical Evaluation %w/w
1 Total Ash 20.21
2 Acid Insoluble Ash 6.10
3 Water Soluble Ash 7.56
4 Loss on Drying 0.64

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Table 4: Extractive values of leaf of shaeranthus
indicus with different solvents.
S. No Sample Extractability (%)
1 Petroleum ether extract 9.96
2 Benzene Extract 1.20
3 Chloroform Extract 0.64
4 Acetone Extract 0.98
5 Methanolic Extract 4.20
6 Ethanol Extract 5.82
7 Aqueous Extract 2.84
1.
Baura Talam, Bodasoram, Bodataram, Chagulnadi,
Ghorkmundi, Globe-thistle, Gorkhumundi, Guroli,
Kamazariyus, Kamdaryus, Mundi, Mundiriki, Murmuriya,
Shosimundi, Thistle, Globe.
Vernacular Names11-13
Tamil : Kottakaranthai
Sans : Mahamundi, Mundi, hapusa
Hindi : Mundi, Gorakh Mundi
Bengali : Mundi, Gorakh Mundi
Gujarati : Mundi, Gorakh Mundi
Telugu : Boddasoramu
Malayalam: Adakkamaniyam
Punjabi : Khamadrus
Macroscopy of the leaf
Morphological studies were done by using simple
microscope to determine the shape, size, taste and odour of
the leaf and sheathing leaf base. Macroscopically the
leaves are simple, alternate, oblong, spatulate, spinous,
surface pubescent, base decurrent forming the wings of the
stem, acute, glandular, hairy and narrowed at the base up
to 5.0x1.5 cm, the leaf margins are coarsely serrate or
dentate. Fresh leaves are dark green in colour and dried
leaves are greenish black colour. The leaves are bitter in
taste with pleasant odour when fresh, the aroma gradually
diminishing on drying and storing.
Microscopical study of the leaf
MATERIALS AND METHODS14
Fresh leaf was used for microscopical examination. The
cut portion of the leaf was first fixed using FAA (Formalin
5ml +Acetic acid 5ml+Ethanol 90ml.). After 24 hours of
fixing, the specimens were dehydrated with graded series
of tertiary butyl alcohol then infiltration by paraffin wax
(58-60o
c). The specimens were cast in to paraffin blocks.
The paraffin embedded specimens were sectioned with the
help of microtome. The sections were stained with
Toluidine blue.
Quantitative microscopy and Physico chemical
parameters
The vein islet number, vein terminal number, stomatal
number, stomatal index were determined on fresh leaves
using standard procedure15-17
. The parameters were done
to evaluate the proceedings of vein islet number, vein
termination number; stomatal number, stomatal index,
total ash, water soluble ash, and acid insoluble ash were
calculated as per Indian Pharmacopoeia18
. Extracts of the
powdered leaf was prepared with different solvents for the
study of extractive value. Fluorescence analysis was also
carried out for the powder and for extract as per standard
procedure19
Powder analysis
Preliminary phytochemical analysis of the powder of the
leaf of S. indicus with different chemical reagents was
carried out microscopically20-21
Extraction of Plant material
For preliminary Phytochemical analysis, extract was
prepared by weighing 1kg of the dried leaf powder were
subjected to hot successive continuous extraction with
different solvents as per the polarity, petroleum ether,
benzene, chloroform, acetone, ethanol, methanol and
finally with aqueous. The extracts were filtered in each
step using Whatman filters paper. The filtrate was
concentrated using a rotary evaporator at low temperature
(40-45
o
C) and pressure. The presence or absence of the
primary and secondary phytoconstituents was detected by
usual prescribed methods22
Thin layer chromatography of Aqueous and Methanol
Extract
Methanolic extract have been reported for the presence of
a novel isoflavone glycoside. 5, 4–dimethoxy-3-
prenylbiochanin –7-O--D-glactoside and the preliminary
phytochemical screening of aqueous and methanolic
extracts were revealed the presence of isoflavone
glycoside. Since an attempt has been made to confirm the
presence of this compound in both the extracts by (Viz
aqueous and methanol) thin layer chromatography using
chloroform: methanol (11:9) as mobile phase and UV light
and Ammonia vapour were used as visualizing agents.
Isolation of volatile oil from the leaves of sphaeranthus
indicus23
The leaf powder was extracted with petroleum ether (40o
–
60o
). The solvent was distilled off. The extracted residue
was subjected to hydro distillation in a volatile oil
estimation apparatus and distillate collected over solvent
ether. The aqueous part was rejected and the ethereal part
was dried over anhydrous sodium sulphate. The solvent
was dried in a weighed conical flask on a water bath at
controlled temperature and kept in vaccum desiccator
overnight and weighed. The yield comes to be 0.01 –
Table 3: Fluorescence analysis of leaf of sphaeranthus indicus.
S. No Sample Colour in Day Light Colour in UV Light
1 Petroleum ether extract Pale Yellow Yellow
2 Benzene Extract Yellow Orange Red
3 Acetone Extract Green Red
4 Chloroform Extract Yellowish green Yellow
5 Methanolic Extract Green Light blue
6 Ethanol Extract Green Dark Green
7 Aqueous Extract Yellow Blue
1.

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Table 6: Physical parameters of oil.
Wt.per
ml(gm/ml)
Refractive index Optical rotation
0.9935 1.5055  Oo
0.02% on fresh weight basis. The isolated oils are
physically and chemically analysed23-26
.
Thin layer Chromatography of volatile oil27-30
It is apparent that silica gel TLC is a choice technique for
the study of essential oils because of its rapidity and
simplicity. With the help of TLC hundreds of oils of
different chemical races have been screened and their
components were identified. Evaluate the essential oil of
this plant by TLC using mobile phases in different ratios
like Toluene: Ethyl acetate (93:7) and (95:5) and Rf values
and colour of the components were recorded. Five
reference standards were used vice Eugenol, Citral,
Geraniol, Ionone and Geranyl acetate to identify these
constituents of this oil and these constituents were
confirmed by co-TLC using the solvent system like
Toluene:Ethylacetate (93:7) and Hexane: Chloroform
(70:30).
Chemical analysis of the essential oil of s.indicus by gas
liquid chromatography 31
Essential oil Isolated from leaves of S.indicus
Reference compounds - Eugenol, Geraniol, Citral, Geranyl
acetate, and ionone
Chromatographic conditions
Stationary phase: Capillary glass column BPX-70
(equivalent to FFAP)30 m long and 0.2 mm in internal
diameter, the inner Surface of which is coated with a layer
of 50% cyano propyl / 50% methyl silicone.
Mobile phase: Nitrogen gas
Flow rate: 25ml / min
Sensitivity: 1
Detector : FID (Flame Ionisation Detector)
Detector temperature: 230o
C
Injector: Septum type with SGE syringe
Injector temperature: 220o
C
Column temperature: 160o
C
Instrument:
Chemitto model gc – 8610, with one packed column and
one capillary column provision, with wichrom software
with data collector.
METHOD
Stabilize the baseline for about 30 minutes with the above
chromatographic conditions. About 1l of Eugenol RS
was injected using SGE Syringe and the chromatogram
was recorded. The same procedure was adopted for other
reference standards viz, citral, ionone, geranyl acetate and
geraniol and their standard chromatograms were recorded.
About 1l of the sample of S.indicus oil was injected and
the sample chromatogram was recorded. The retention
time was determined for the sample and the standard. The
peak area of the standard and sample were determined. The
composition of the components of the Essential oil was
calculated using the formula.
Percentage Composition of the components in the sample
= Area of the sample peak
------------------------------ ×100
Area of the standard peak
RESULTS
Anatomy of the Leaf
Leaf
The leaf has distinct midrib and uniformly thick lamina.
The midrib is Plano convex in sectional view, the adaxial
side is more or less flat and the abaxial side is broad and
hemispherical (Fig 1). The epidermis is thin and consists
of squarish or elliptical cells with thin cuticle. There is a
single top-shaped, collateral vascular bundle; the bundle is
surrounded by parenchymatous ground tissue. The
vascular bundle has four or five parallel rows of xylem
elements and a thick are of phloem elements (Fig 2). No
sclerenchyma cells are seen in the vascular bundle.
Lamina
The lamina has distinct, fairly thick epidermal layers
which bear dense trichomes. The abaxial epidermis is
Table 5: Preliminary phytochemical tests for drug powder and various extracts of leaf of sphaeranthus indicus.
S.No
Test
Drug
Powder
Petroleum
Ether
Extract
Benzene
Extract
Chlorofor
mExtract
Acetone
Extract
Methanol
Extract
Ethanol
Extract
Aqueous
Extract
1 Sterols + + + + + + + -
2 Terpenoids + + + + + + + -
3 Carbohydrates + - - - + + + +
4 Flavanoids + - - - + + + +
5 Proteins - - - - - - - -
6 Alkaloids - - - - - - - -
7 Glycosides - - - - - - - -
8 Saponins - - - - - - - -
9 Tannins + - - - + + + +
10 Mucilages - - - - - - - -
11 Volatile Oil + - - - - - - -
+ indicates positive reaction, -indicates negative reaction.
Table 7: Chemical analysis of oil.
Acid Value Ester value
5.8 75.8

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stomatiferous. The mesophyll tissue is consists of several
layers of cubical or vertically oblong cells; A few layers of
adaxial mesophyl cells appear vertically oblong palisade –
like cells (Fig 3 and 4). The spongy mesophyll tissue is
aerenchymatous and consists of lobed cells.
Epidermal Trichomes (Fig 5, 6)
The epidermal trichomes are characteristic. There are two
types of trichomes on the leaf. One is nonglandular and is
multicellular, uniseriate, unbranched and whip like with
dilated basal cell (Fig 6). The other trichome is glandular
and multicellular and biseriate. The glandular trichome
has two rows of vertically oblong, thin walled cells arising
from dilated basal epidermal cells. (Fig 5) At the terminal
part the cells become shorter to rectangular shape and
finally at the summit are two hemispherical cells. The
summit cells are glandular with dense cytoplasm and
prominent nuclei. (Fig 6)
Venation Pattern: (Fig 7)
The vein islets are distinct, they vary in shape and size;
generally they are rectangular to polygonal. The vein
terminations are mostly single per islet; they are simple or
branched once (Fig 7). In paradermal sections, the lateral
veins and veinlets have small continuous sheath cells (Fig
8).
Stomata (Fig 9)
Table 8: T.L.C of volatile oil.
Mobile phase Adsorbent Visualizing Agent
Rf values of the
spots
Colour
Toluene : Ethyl
acetate (93:7)-(Fig
14)
Silicagel -G (activated at
110o
for 30 mts)
5% Vanillin sulphuric
acid
(i) 0.34 Green
(ii) 0.4 Blue
(iii) 0.46 Greenish Blue
(iv) 0.7 Reddish brown
(v) 0.71 Pink
(vi) 0.73 Violet
(vii) 0.84 Light Pink
(viii) 0.92 Greenish Black
Toluene: Ethyl
acetate (95:5)
Fig (15)
Silica gel- G (activated at
110o
for 30mts)
5% Vanillin sulphuric
acid
0.1 Rose
0.17 Violet
0.6 Pink
0.95 Blue
Table 9: Co- T.L.C of volatile oil.
Mobile phase Adsorbent
Visualizing
Agent
Standard
Sample
Rf Values
ColourName of the
Standard
Rf Values
Toluene: Ethyl
acetate (93:7)
(Fig 16)
Silica gel-G
(activated at
110o for 30mts)
5% vanillin
sulphuric acid
Eugenol 0.7 0.7
Reddish
brown
Geraniol 0.84 0.84 Light pink
Ionone 0.46 0.46
Greenish
blue
Geranyl
Acetate
0.5 -
Greenish
black
Hexane:
chloroform
(70:30) (Fig 17)
Silica gel -G UV light Citral 0.92 0.92 Bluish green
Table 10: GLC analysis of volatile oil
Name of the
Reference standard
Retention Time Area of the Peak Percentage
composition of
components in the
sample
Standard Sample Standard Sample
Eugenol 3.86 3.76 29408069 212022 0.72
Citral 1.88 1.98 8257500 147583 1.7
Geraniol 1.62 1.74 11529021 306969 2.6
Ionone 2.54 2.68 32005243 4413481 13.78
Geranylacetate 1.44 - 15676144 - -

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Stomata are exclusively anomocytic; the guard cells are
not surrounded by subsidiary cells distinctly differently
from the neighbouring epidermal cells. The anticlinal walls
of the epidermal cells are highly wavy and the epidermals
become much lobed. The cell walls are thin; cuticular
striations are not evident.
Figure 4: T.S of lamina with trichome Figure 5: Glandular trichome enlarged
Figure 6: Trichome morphology Figure 7: Venation patteren
Figure 8: Paradermal section of the leaf Figure 9: Stomata
Figure 10; T.S of petiole Figure 11: T.S of petiole enlarged

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Figure 12: T.L.C of aqueous extract Figure 13: T.L.C of methanolic extract
Figure 14: T.L.C of volatile oil (1) Figure 15: T.L.C of volatile oil (2)
Figure 16: co T.L.C of volatile oil Figure 17: co- T.L.C of volatile oil
Microscopy of petiole (fig 10,11)
The petiole is circular, even and smooth in cross-sectional
view. It has outer aerenchymatous ground tissue, a circular
vascular cylinder and central parenchymatous ground
tissue. (Fig 10) These are distinct their epidermal layer and
one or two subepidermal, compact parenchyma cells; the
aerenchymatous zone consists of 2 or 3 layers of wide air-
chambers separated from each other by uniseriate
partitions. (Fig 11) The cells of the aerenchyma are
angular, compact and thin walled. The vascular cylinder
has several wedge-shaped vascular bundles forming a
circle with interfascicular parenchymatous gap. (Fig 11)
The vascular bundles are collateral; the xylem elements are
in 3-5 radial rows; phloem occurs as thick mass on the
outer part of the xylem.
Powder Microscopy
The organoleptic evaluation of the leaf powder shows that
it was coarse, green with aromatic odour having slightly
bitter taste. Fragments of parenchyma cells, aerenchyma
cells and collenchymas cells. Glandular trichomes have
two rows of vertically oblong thin walled cells. Non
glandular trichomes are multicellular, uniseriate,
unbranched, whip like with dilated basal vessel. When
stained with toulidine and anomocytic stomata were
observed when stained with aniline blue and vascular
bundles were observed, when stained with phluroglucinol
and concentrated hydrochloric acid.
Quantitative microscopy
The quantitative microscopy such as vein- islet number,
vein- terminal number, stomatal number and stomatal
index were determined and the results were tabulated.
(Table 1)

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GLC of the oil of S,indcus GLC of eugenol
GLC of citral GLC of geraniol
GLC of ionone GLC of geranyl acetate

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Physico chemical features
The powdered drug was evaluated for its physico-chemical
parameters like total ash values, acid insoluble ash, water
soluble ash and loss on drying, and the results were
tabulated (Table 2).
Fluorescence analysis of the extracts
The extracts were prepared as per their polarity in hot
successive extraction technique, and they were treated with
reagents and the colour changes were observed under Ultra
Violet light and the results were tabulated (Table 3).
Extractive values
The extracts were prepared according to the polarity and
they were concentrated and their values were calculated
with reference to air dried drug and the results were
tabulated (Table 4).
Preliminary phytochemical analysis
The leaf powder and various extracts such as petroleum
ether extract, benzene extract, chloroform extract, ethanol
extract and aqueous extract were subjected to preliminary
phytochemical screening for their presence or absence of
the constituents and the results were tabulated (Table 5).
Thin layer chromatography of Aqueous and Methanolic
Extract
A yellow colour spot was obtained with both the extracts
indicate the presence of isoflavone glycoside. (Fig 12, 13)
The phytochemical tests and TLC studies reveals the
presence of Isoflavone compound in both the extracts
Analysis of volatile oil
Physical analysis
Colour : Very deep sherry red
Odour : Aromatic and pleasant
Taste : Bitter
Solubility : Soluble in water, alcohol, acetone, chloroform,
Toluene, benzene and ether
The weights per ml, refractive index and optical rotation
are some important distinctive criteria for the oils when
where determined and tabulated (Table 6)
Chemical Analysis
The essential oil is analysed chemically by its acid value,
and ester value which were determined and tabulated.
(Table 7)
Thin layer Chromatography of volatile oil
The essential oil of this plant confirmed by TLC using
mobile phases in different ratios and Rf values and colour
of the components were recorded and tabulated. (Table 8)
Five reference standards were used vice Eugenol, Citral,
Geraniol, Ionone and Geranyl acetate to identify the
constituents of this oil and the constituents were confirmed
by co-TLC.
Analysis of oil of Sphaeranthus indicus by GLC
Essential oil have been analysed by GLC and their
components were identified and quantified. Five standards
viz Eugenol, geraniol, citral, Geranyl acetate, and ionone
were used and the standard chromatograms were recorded.
The sample chromatogram also recorded with the oil of
Sphaeranthus indicus.The various parameters of the GLC
of the oil such as retention time and area of the peak were
considered for standards and sample. The retention time
for the reference standard geraniol was 1.62 minutes
corresponding to the area of the peak 11529021, where as
in the sample the retention time for Geraniol was 1.74
minutes corresponding to the area of the peak 306969 and
the percentage of Geraniol in the sample was calculated as
2.6 The retention time for the reference standard Eugenol
was 3.86 minutes corresponding to the area of the peak
29408069 where as in the sample retention time for
Eugenol was 3.76 minutes corresponding to the area of the
peak 212022 and the percentage of Eugenol in the sample
was calculated as 0.72. The retention time for the
reference standard Citral was 1.88 minutes corresponding
to the area of the peak 8257500 where as in the sample
retention time for Citral was 1.98 minutes corresponding
to the area of the peak 147583 and the percentage of citral
in the sample was calculated as 1.7, The retention time for
the reference standard ionone was 2.54 minutes,
corresponding to the area of the peak 32005243, where as
in the sample retention time for ionone was 2.68 minutes
corresponding to the area of the peak 4413481 and the
percentage of ionone in the sample was calculated as
13.78. The retention time for the reference standard
Geranyl acetate was 1.44 minutes corresponding to the
area of the peak 15676144 whereas the sample retention
time did not correlate the standard retention time. So it did
not contain geranyl acetate. The retention time and area of
the peaks are tabulated. (Table 10)
DISCUSSION
Our study has focused on examining Pharmacognostic and
Preliminary phytochemical and T.L.C, G.L.C studies of
Sphaeranthus indicus leaves. Normalization of the
macroscopic and microscopic characteristics of the
S,indicus. Drug remains essential in other to identify and
avoid falsification. The leaf has distinct midrib and thick,
soft lamina. The midrib is plano-convex in cross-sectional
view with single top-shaped collateral vascular bundle
surrounded by parenchymatous cells. No sclerenchyma
cells are seen in the vascular bundle. The lamina is
dorsiventral; however, the mesophyll tissue is not well
differentiated into palisade and spongy tissues.
Characteristic epidermal trichomes are abundant on the
leaf. Some of the trichomes are covering-type and are
multicellular, uniseriate, unbranched and whip-like others
are biseriate, broad, unbranched, conical with vertically
oblong cells and a few tiers of apical glandular cells.
Stomata are anomocytic; anticlinal walls of the epidermal
cells are highly wavy. Vein islets are distinct, with one,
simple or branched vein terminations. Petiole is circular in
sectional view with aerenchymatous outer ground tissue,
broad central tissue and is open ring of discrete collateral
vascular bundles.Organoleptic characteristics are
important in drugs because they play a role in the detection
of adulterated or substituted drugs32
.Thus leaves dark
green in colour, emit a very fragrant and aromatic
mintyodor when bruised. The powdery appearance of the
crushed leaves, with a coarse texture. The micrograph
performed on the powder has highlighted a number of
characteristic elements namely: the polygonal, wavy
epidermal cells, the anomocytic type of
stomata,Glandular,Nonglandular trichomes, are diagnostic
substances for drugs of plant origin. These diagnostic

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IJPPR, Volume 10, Issue 1: January 2018 Page 61
elements are consistent with botanical standards and WHO
guidelines33-34
. The study of physicochemical parameters
such as moisture content and ash values are useful as it
determines the physiological and nonphysiological state of
ash, this will help to determine the possibility of microbial
growth and lastly contaminant or impurities. The moisture
content of the drug studied had a rate of 0.68 ± 0,1, which
is below 10%. This result comply with the standards
established by the International Pharmacopoeia, because
this water content rate, prevent oxidation reactions,
fermentation and give less chance to microbial growth and
contamination in drugs35
. Therefore, for proper
conservation of drugs made from the leaves of S.hirta., it
would be desirable to use those whose water content is less
than or equal to 10%..The determination of total ash gave
us a rate of 20.21 ± 0.03. This value indicates the level of
minerals in drugs. Insoluble ash in hydrochloric acid gave
a rate of 6.10 ± 0,02. Indeed, the ash insoluble in
hydrochloric acid tells us about the contamination of the
drug by siliceous elements36
. This result is in agreement
with Srikanth et al.37
who found rate of 0.97% and 0.5%
respectively. The maximum extractive value was found in
distilled water (12.84%) followed by Petroleum ether
(9.96%), Ethanol (5.82%) methanol (4.20%) Benzene
(1.20%), Acetone (0.98%) Chloroform (0.64%). All the
extracts of the drug was subjected to different tests for
detecting the presence of various phytoconstituents present
in the drug, which revealed the presence of
sterols,terpenoids, flavanoids, and tannins. Preliminary
phytochemical analysis indicated a high percentage of
quercetine and flavonoids and this may be one of the
reasons behind the hypolipidemic activity of the plant.
plant. TLC profile of aqueous and methanolic extracts
showed yellow colour spots under UV, indicates the
presence of isoflavonoids, T.L.C analysis of volatile oil of
S.indicus showed seven spots, these were compared with
co TLC, it indicates that the presence of Eugenol, Geraniol,
Ionone, and Citral. GLC analysis of volatile oil obtained
from S.indicus indicates that the presence the above
volatile substances. These parameters, which are being
reported for the first time in this plant, are significant
towards establishing the pharmacognostic standards for
future identification and authentication of genuine plant
material. Though Sphaeranthus indicus is a weed, it is a
highly reputed drug used in Ayurveda. Barring the
anatomical details and preliminary phytochemical
screening, rest of the pharmacognostical parameters, gives
us the clue that it can be cashed economically as well to
improve the standard of health in the developing countries.
CONCLUSION
Microscopic method is one of the simplest and cheapest
methods to start with, for establishing the correct identity
of the source materials. Sphaeranthus indicus L. is
often confused with S. amaranthoides and other members
of Asteracea. When the specimens are in fragmentary
condition, to identify the crude fragmentary plant
materials, anatomical characters are often helpful, this
research paper covers an extensive study on the leaves of
Sphaeranthus indicus. The Pharmacognostic,
Phytochemical profile including preliminary
phytochemical tests, TLC and GLC analysis of essential
oil obtained from the leaves. Pharmacognostic parameters
have been determined for leaf in order to substantiate and
identify the plant for future work. It gives us the clue that
it can be cashed economically as well to improve the
standard of health in the developing countries.
ACKNOWLEDGEMENT
The authors are thankful to the Director and Principal of
Sanjo College of Pharmaceutical studies, Vellapara,
Palakkad for providing facilities to carry out the present
research work and also thankful to Baid mehtha
analytical lab, Chennai.
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